Acyl phosphonate inhibitors of β-lactamases

ABSTRACT

The invention provides novel β-lactamase inhibitors, which are structurally unrelated to the natural product and semi-synthetic β-lactamase inhibitors presently available, and which do not possess a β-lactam pharmacophore. These new inhibitors are fully synthetic, allowing ready access to a wide variety of structurally related analogs. Certain embodiments of these new inhibitors also bind bacterial DD-peptidases, thus potentially acting both as β-lactamase inhibitors and as antibiotics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/582,255, filed on Jun. 22, 2000, which is a U.S. nationalstage application of international patent application No.PCT/US98/27518, filed on Dec. 23, 1998, which in turn claims priorityfrom U.S. provisional patent application serial No. 60/068,837, filed onDec. 24, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to bacterial antibiotic resistance. Moreparticularly, the invention relates to compositions and methods forovercoming bacterial antibiotic resistance.

2. Background of the Invention

Bacterial antibiotic resistance has become one of the most importantthreats to modern health care. Cohen, Science 257:1051-1055 (1992)discloses that infections caused by resistant bacteria frequently resultin longer hospital stays, higher mortality and increased cost oftreatment. Neu, Science 257:1064-1073 (1992) discloses that the need fornew antibiotics will continue to escalate because bacteria have aremarkable ability to develop resistance to new agents rendering themquickly ineffective.

The present crisis has prompted various efforts to elucidate themechanisms responsible for bacterial resistance. Coulton et al. Progressin Medicinal Chemistry 31:297-349 (1994) teach that the widespread useof penicillins and cephalosporins has resulted in the emergence ofβ-lactamases, a family of bacterial enzymes that catalyze the hydrolysisof the β-lactam ring common to presently used antibiotics. Morerecently, Dudley, Pharmacotherapy 15: 9S14S (1995) has disclosed thatresistance mediated by β-lactamases is a critical aspect at the core ofthe development of bacterial antibiotic resistance.

Attempts to address this problem through the development of β-lactamaseinhibitors have had limited success. Sutherland, Trends Pharmacol Sci12: 227-232 (1991) discusses the development of the first clinicallyuseful β-lactamase inhibitor, clavulanic acid, which is a metabolite ofStreptomyces clavuligerus. Coulton et al. (supra) disclose two othersuch semi-synthetic inhibitors, sulbactam and tazobactam presentlyavailable. Coulton et al. (supra) also teach that in combination withβ-lactamase-susceptible antibiotics, β-lactamase inhibitors preventantibiotic inactivation by β-lactamase enzymes, thereby producing asynergistic effect against β-lactamase producing bacteria.

Rahil and Pratt, Biochem. J. 275: 793-795 (1991), and Li et al., Bioorg.Med. Chem. 5: 1783-1788 (1997) teach that β-lactamase enzymes areinhibited by phosphonate monoesters. Song and Kluger, Bioorg. Med. Chem.Lett., 4, 1225-1228 (1994), teaches that E. Coli RTEM β-lactamase isinhibited by benzylpenicillin methyl phosphate.

Laird and Spence, J. C. S. Perkin Trans. II 1434 (1973), Kazlauskas andWhitesides, J. Org. Chem. 50: 1069-1076 (1985), Chantrenne, Compte.Rend. Trav. Lab. Carlsberg Ser. Chim. 26: 297 (1948), and Maracek andGriffith, J. Am. Chem. Soc. 92: 917-921 (1970), report synthesis andsolvolysis studies of acyl phosphate and acyl phosphonate compounds.Kluger et al., Can. J. Chem. 74: 2395-2400 discloses that aminoacylphosphates are useful as biomimetically activated amino acids.

The availability of only a few compounds however, is insufficient tocounter the constantly increasing diversity of β-lactamases for which avariety of novel and distinct inhibitors has become a necessity. Thereis, therefore, a need for the ability to identify new β-lactamaseinhibitors. The development of fully synthetic inhibitors would greatlyfacilitate meeting this need. Ideally, certain embodiments of suchinhibitors would also bind bacterial DD-peptidases, thus potentiallyacting both as β-lactamase inhibitors and as antibiotic agents.

BRIEF SUMMARY OF THE INVENTION

The invention provides novel β-lactamase inhibitors, which arestructurally unrelated to the natural product and semi-syntheticβ-lactamase inhibitors presently available, and which do not possessβ-lactam pharmacophore. These new inhibitors are preferably fullysynthetic, allowing ready access to a wide variety of structurallyrelated analogs. Certain embodiments of these new inhibitors also bindbacterial DD-peptidases, and thus potentially act both as β-lactamaseinhibitors and as antibiotics.

In a first aspect, the invention provides novel acyl phosphate and acylphosphonate β-lactamase inhibitors. Preferably, such inhibitors have thegeneral mixed anhydride structure of Formula I:

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided, however, that when Y is Z, then Z is notphosphonyl; and further provided that when Y is OZ and Z is phenyl, thenX is not methyl or phenyl; when Y is OZ and Z is alkyl or adenosyl, thenX is not α-aminoalkyl; and when Y is OZ and Z is benzoyl, then X is notphenyl.

In certain preferred embodiments, the mixed anhydride is an acylphosphate, and Y is thus OZ. In certain other preferred embodiments, themixed anhydride is an acyl phosphonate, and Y is thus Z.

In certain preferred embodiments, Y and X are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted; provided that X is not phenylethene.

In a second aspect, the invention provides pharmaceutical compositionscomprising an acyl phosphate or acyl phosphonate β-lactamase inhibitorand a pharmaceutically acceptable carrier, excipient, or diluent.Preferably, such inhibitors have the general mixed anhydride structure(I):

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

In certain preferred embodiments, Y and X are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted.

In a third aspect, the invention provides methods for inhibiting invitro or in vivo β-lactamase activity, such methods comprisingadministering an acyl phosphate or acyl phosphonate β-lactamaseinhibitor. Preferably, such inhibitors have the general mixed anhydridestructure of Formula I:

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

In certain preferred embodiments, X and Y are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted

In a fourth aspect, the invention provides a method for inhibitingbacterial growth, the method comprising administering an acyl phosphateor acyl phosphonate β-lactamase inhibitor. Preferably, such inhibitorshave the general mixed anhydride structure of Formula I:

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

In certain preferred embodiments, X and Y are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted

In one embodiment of such a method, β-lactamase inhibitor according tothe invention is co-administered with an antibiotic. In anotherembodiment the β-lactamase inhibitor according to the invention hasantibiotic activity, and can thus either be administered alone or beco-administered with another antibiotic agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to bacterial antibiotic resistance. Moreparticularly, the invention relates to compositions and methods forovercoming bacterial antibiotic resistance.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. The issuedpatents, applications, and references that are cited herein are herebyincorporated by reference to the same extent as if each was specificallyand individually indicated to be incorporated by reference. In the caseof inconsistencies, the present disclosure will prevail.

The invention provides novel β-lactamase inhibitors, which arestructurally unrelated to the natural product and semi-syntheticβ-lactamase inhibitors presently available, and which do not possess aβ-lactam pharmacophore. These new inhibitors are preferably fullysynthetic, allowing ready access to a wide variety of structurallyrelated analogs. Certain embodiments of these new inhibitors also bindbacterial DD-peptidases, and thus potentially act both as β-lactamaseinhibitors and as antibiotics.

For purposes of the present invention, the following definitions will beused:

Definitions

As used herein, the term “β-lactamase” denotes a protein capable ofinactivation of a β-lactam antibiotic. In one preferred embodiment, theβ-lactamase is an enzyme which catalyzes the hydrolysis of the β-lactamring of a β-lactam antibiotic. In certain preferred embodiments, theβ-lactamase is microbial. In certain preferred embodiments, theβ-lactamase is a serine β-lactamase. In certain other preferredembodiments, the β-lactamase is a zinc β-lactamase. The terms “class A”,“class B”, “class C”, and “class D” β-lactamases are understood by thoseskilled in the art and can be found described in Waley, The Chemistry ofβ-Lactamase, Page Ed., Chapman & Hall, London, (1992) 198-228. Inparticularly preferred embodiments, the β-lactamase is class Cβ-lactamase of Enterobacter cloacae P99 (hereinafter P99 β-lactamase),or class A β-lactamase of the TEM-2 plasmid (hereinafter TEMβ-lactamase).

As used herein, the term “β-lactamase inhibitor” is used to identify acompound having a structure as defined herein, which is capable ofinhibiting β-lactamase activity. Inhibiting β-lactamase activity meansinhibiting the activity of a class A, B, C, or class D β-lactamase.Preferably, such inhibition should be at a 50% inhibition concentrationbelow 100 micrograms/mL, more preferably below 30 micrograms/mL and mostpreferably below 10 micrograms/mL.

In some embodiments of the invention, the β-lactamase inhibitor is alsocapable of acting as an antibiotic, for example, by inhibiting bacterialcell-wall cross-linking enzymes. Thus, the term β-lactamase inhibitor isintended to encompass such dual-acting inhibitors. In certain preferredembodiments, the β-lactamase inhibitor is capable of inhibitingD-alanyl-D-alanine-carboxy-peptidases/transpeptidases (hereinafterDD-peptidases). The term “DD-peptidase” is used in its usual sense todenote penicillin-binding proteins involved in bacterial cell wallbiosynthesis (e.g., Ghysen, Prospect. Biotechnol. 128:67-95 (1987)). Incertain particularly preferred embodiments, theD-alanyl-D-alanine-carboxy-peptidases/transpeptidase inhibited is theStreptomyces R61 DD-peptidase.

The term “alkyl” as employed herein refers to straight and branchedchain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8carbon atoms, which may be optionally substituted with one, two or threesubstituents. Unless otherwise specified, the alkyl group may besaturated, unsaturated, or partially unsaturated. As used herein,therefore, the term “alkyl” is specifically intended to include alkenyland alkynyl groups, as well as saturated alkyl groups. Preferred alkylgroups include, without limitation, methyl, ethyl, propyl, isopropyl,butyl, tert-butyl, isobutyl, pentyl, hexyl, vinyl, allyl, isobutenyl,ethynyl, and propynyl.

The term “alkylene” as employed herein refers to saturated, unsaturated,and partially unsaturated groups having from 1 to 8 carbon atoms,preferably 1-6 carbon atoms, more preferably 1-4 carbon atoms, and mostpreferably 1-2 carbon atoms, positioned between and connecting two othersubstituents. Preferred alkylene groups include, without limitation,methylene, ethylene, and ethene. For purposes of the invention, analkylene group preferably refers to a portion of a cyclic structure.

As employed herein, a “substituted” alkyl, cycloalkyl, aryl, orheterocyclic group is one having between one and about four, preferablybetween one and about three, more preferably one or two, non-hydrogensubstituents. Suitable substituents include, without limitation, halo,hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, amino,alkylcarboxamido, arylcarboxamido, aminoalkyl, alkoxycarbonyl, carboxy,hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, cyano, andalkylaminocarbonyl groups.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12,preferably 3 to 8, more preferably 3 to 6 carbons, wherein one or tworing positions may be substituted with an oxo group, and wherein thecycloalkyl group additionally may be optionally substituted. Preferredcycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexanone,cycloheptyl, and cyclooctyl. A “cycloalkylene” group is a cycloalkylgroup positioned between and connecting two other substituents.Preferred cycloalkylene groups include, without limitation,cyclohexylene, cyclopentylene, and cyclobutylene.

An “aryl” group is a C₆-C₁₄ aromatic moiety comprising one to threearomatic rings, which may be optionally substituted. Preferably, thearyl group is a C₆-C₁₀ aryl group. Preferred aryl groups include,without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An“aralkyl” or “arylalkyl” group comprises an aryl group covalently linkedto an alkyl group, either of which may independently be optionallysubstituted or unsubstituted. Preferably, the aralkyl group is(C₆₋₁₀)ar(C₁₋₆)alkyl, including, without limitation, benzyl, phenethyl,and naphthylmethyl. An “alkaryl” or “alkylaryl” group is an aryl grouphaving one or more alkyl substituents. Examples of alkaryl groupsinclude, without limitation, tolyl, xylyl, mesityl, ethylphenyl, andmethylnaphthyl.

An “arylene” group is a C₆₋₁₀ aryl group positioned between andconnecting two other substituents. The arylene group may be optionallysubstituted. A non-limiting example of an arylene group is phenylene.For purposes of the invention, the arylene group preferably constitutesone ring of a fused bicyclic or tricyclic ring system.

A “heterocyclic” group or radical is a ring structure having from about3 to about 8 atoms, wherein one or more atoms are selected from thegroup consisting of N, O, and S. The heterocyclic group may beoptionally substituted on carbon with oxo or with one of thesubstituents listed above. The heterocyclic group may also independentlybe substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl,alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl,aralkoxycarbonyl, or on sulfur with oxo or lower alkyl. Preferredheterocyclic groups include, without limitation, epoxy, aziridinyl,tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl,thiazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholino.

In certain preferred embodiments, the heterocyclic group is a heteroarylgroup. As used herein, the term “heteroaryl” refers to groups having 5to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or14 π electrons shared in a cyclic array; and having, in addition tocarbon atoms, between one and about three heteroatoms selected from thegroup consisting of N, O, and S. Preferred heteroaryl groups include,without limitation, thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl,quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, thiazolyl,and isoxazolyl. In certain other preferred embodiments, a heterocyclicgroup is fused to an aryl or heteroaryl group. Examples of such fusedheterocyles include, without limitation, tetrahydroquinoline anddihydrobenzofuran.

A “heteroarylene” group is a heteroaryl group positioned between andconnecting two other groups. The heteroarylene group may be optionallysubstituted. For purposes of the invention, the heteroarylene grouppreferably forms one ring of a fused bicyclic or tricyclic ring system.

The term “halogen” or “halo” as employed herein refers to chlorine,bromine, fluorine, or iodine.

As herein employed, the term “acyl” refers to an alkylcarbonyl orarylcarbonyl substituent, wherein the alkyl or aryl portion may beoptionally substituted.

The term “amido” as employed herein refers to a formylamino,alkylcarbonylamino, or arylcarbonylamino group. The term “amino” ismeant to include NH₂, alkylamino, arylamino, and cyclic amino groups.

The term “phosphate” refers to groups in which there are four oxygenatoms around a phosphorous atom. The term “phosphonate” refers to groupsin which there are three oxygen atoms around a phosphorous atom. Theterm “phosph(on)ate”, as used herein refers generally to either aphosphate or a phosphonate. The term “phosphonyl” refers to a radical inwhich there are three oxygen atoms around a phosphorous atom. Thephosphonyl radical may be attached to carbon to form a phosphonate groupor to oxygen to form a phosphate group.

In a first aspect, the invention provides novel acyl phosphate and acylphosphonate β-lactamase inhibitors. Preferably, such inhibitors have thegeneral mixed anhydride structure (I):

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided, however, that when Y is Z, then Z is notphosphonyl; and further provided that when Y is OZ and Z is phenyl, thenX is not methyl or phenyl; when Y is OZ and Z is alkyl or adenosyl, thenX is not α-aminoalkyl; and when Y is OZ and Z is benzoyl, then X is notphenyl.

In certain preferred embodiments, the mixed anhydride is an acylphosphate, and Y is thus OZ. In certain other preferred embodiments, themixed anhydride is an acyl phosphonate, and Y is thus Z. For purposes ofthe present invention, the term “acyl phosph(on)ate” will be used torefer generally to either acyl phosphates or acyl phoshonates.

Z is preferably selected from the group consisting of alkyl, preferablyC₁₋₆ alkyl; aryl, preferably C₆₋₁₄ aryl, more preferably C₆₋₁₀ aryl,most preferably phenyl or substituted phenyl; aralkyl, preferably(C₆₋₁₀)ar(C₁₋₆)alkyl, more preferably (C₆₋₁₀)ar(C₁₋₃)alkyl, mostpreferably benzyl or naphthylmethyl; acyl, preferably acetyl, benzoyl,or substituted benzoyl; heterocyclic radical, preferably heteroaryl orfused heteroaryl; and phosphonyl.

X is selected from the group consisting of alkyl, preferably C₁₋₆ alkyl;aryl, preferably C₆₋₁₄ aryl, more preferably C₆₋₁₀ aryl, most preferablyphenyl or substituted phenyl; aralkyl, preferably (C₆₋₁₀)ar(C₁₋₆)alkyl,more preferably (C₆₋₁₀)ar(C₁₋₃)alkyl, most preferably benzyl ornaphthylmethyl; and heterocyclic radical, preferably heteroaryl or fusedheteroaryl. In some preferred embodiments, X is aminoalkyl. In theseembodiments, X together with the adjacent —C(O)—O— preferably forms anα-amino acid, more preferably a naturally occurring α-amino acid.

Any of the alkyl, aryl, or heteroaryl groups in X or Z may be optionallysubstituted with one to three, preferably one or two, substituentsselected from the group consisting of halo, hydroxy, nitro, haloalkyl,alkyl, alkaryl, aryl, aralkyl, alkoxy, amino, alkylcarboxamido,arylcarboxamido, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, cyano, and alkylaminocarbonyl. Morepreferably, an aryl or heteroaryl group in X or Z is optionallysubstituted with one or two substituents independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ alkoxy, C₆₋₁₀ aryl, and nitro.

In certain preferred embodiments, Y and X are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene, and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted; provided that X is not phenylethene. Incertain preferred embodiments, X is a fused carbocyclic, heterocyclic,aromatic, or heteroaromatic ring. In one such preferred embodiment, X isphenylene, giving the structure of Formula III:

wherein Y is O, or alkylene.

In neutral aqueous solution, most compounds of the invention aresurprisingly stable, with half-lives for spontaneous hydrolysis ofbetween 0.6 and 80 days. Compounds with electron-withdrawing acylsubstituents, such as compound XXIII, or with aliphatic acyl groups,such as compound X, exhibit shorter half-lives. This pattern ofreactivity suggests that attack by water/hydroxide occurs at the acylrather than the phosphyl center. Compounds XXXII and XXIII are morerapidly hydrolyzed.

The acyl phosph(on)ates of the invention are both substrates andirreversible inhibitors of β-lactamases. While not wishing to be boundby theory, the inventors have found that turnover, as a substrate,involves fast acylation followed by slower deacylation, whereasirreversible inactivation involves phosphylation. In general, acylationis far more rapid than phosphylation, and is enhanced by hydrophobicgroups in both the acyl and leaving group moieties of the acylphosph(on)ate. Acylation results in the formation of a tight acyl-enzymecomplex, giving rise to reversible enzyme inhibition.

The compounds according to the invention are useful as β-lactamaseinhibitors. In certain preferred embodiments, the compounds of theinvention also have utility as antibiotic agents. Furthermore, theinhibitors of the invention are also useful as probes for elucidatingand identifying mechanisms responsible for bacterial antibioticresistance and for evaluating the effect of administering agents toovercome such resistance concomitantly with antibiotic treatments.

Nonlimiting examples of certain preferred embodiments according to theinvention appear in Table 1. These examples are shown as salts. It wouldbe evident to one skilled in the art that the compounds of FormulaeI-III can exist in conjugate acid, conjugate base, or salt form. Thedisclosure of compounds, compositions, and methods contained herein is,in each instance, expressly intended to include all such forms.

TABLE 1 IV

V

VI

VII

VIII

IX

X

XI

XII

XVII

XVIII

XIX

XX

XXI

XXII

XXIII

XXIV

XXV

XXVI

XXVII

XXVIII

XXIX

XXX

XXXI

XXXII

XXXIII

In a second aspect, the invention provides pharmaceutical compositionscomprising an acyl phosphate or acyl phosphonate β-lactamase inhibitorand a pharmaceutically acceptable carrier, excipient, or diluent.Preferably, such inhibitors have the general mixed anhydride structure(I):

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

Preferred values for X, Y, and Z according to this aspect of theinvention are as described above for the first aspect.

In certain preferred embodiments, Y and X are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted. In certain preferred embodiments, X is afused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. Inone such preferred embodiment, X is phenylene, giving the structure ofFormula III:

wherein Y is O or alkylene.

The characteristics of the carrier, excipient, or diluent will depend onthe route of administration. As used herein, the term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The term “physiologically acceptable” refers to a non-toxic materialthat is compatible with a biological system such as a cell, cellculture, tissue, or organism. Preferably, the biological system is aliving organism, more preferably a mammal, most preferably a human. Thuscompositions and methods according to the invention, may contain, inaddition to the inhibitor, diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.

In certain preferred embodiments, the pharmaceutical compositionsaccording to this aspect of the invention additionally comprise anantibiotic agent. In particularly preferred embodiments, the antibioticagent is a β-lactam antibiotic. The pharmaceutical composition of theinvention may also contain other active factors and/or agents whichenhance inhibition of β-lactamase and/or DD-peptidases.

The term “antibiotic” is used herein to describe a composition whichdecreases the viability or which inhibits the growth or reproduction ofmicroorganisms. As used in this disclosure, an antibiotic is furtherintended to include an antimicrobial, bacteriostatic, or bactericidalagent. Non-limiting examples of antibiotics useful according to thisaspect of the invention include penicillins, cephalosporins,aminoglycosides, sulfonamides, macrolides, tetracyclins, lincosides,quinolones, chloramphenicol, vancomycin, metronidazole, rifampin,isoniazid, spectinomycin, trimethoprim, sulfamethoxazole, and others.The term “β-lactam antibiotic” is used to designate compounds withantibiotic properties containing a β-lactam functionality. Non-limitingexamples of β-lactam antibiotics useful according to this aspect of theinvention include penicillins, cephalosporins, penems, carbapenems, andmonobactams.

In certain preferred embodiments, the second aspect of the inventionprovides pharmaceutical compositions for use in the methods of theinvention.

In a third aspect, the invention provides methods for inhibiting invitro or in vivo β-lactamase activity, such methods comprisingadministering an acyl phosphate or acyl phosphonate β-lactamaseinhibitor. Preferably, such inhibitors have the general mixed anhydridestructure of Formula I:

or salts thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

Preferred values for X, Y, and Z according to this aspect of theinvention are as described above for the first aspect.

In certain preferred embodiments, X and Y are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted. In certain preferred embodiments, X is afused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. Inone such preferred embodiment, X is phenylene, giving the structure ofFormula III:

wherein Y is O or alkylene.

Non-limiting examples of particularly preferred inhibitors to be usedaccording to this aspect of the invention are shown in Table 2.

TABLE 2 IV

V

VI

VII

VIII

IX

X

XI

XII

XIII

XIV

XV

XVI

XVII

XVIII

XIX

XX

XXI

XXII

XXIII

XXIV

XXV

XXVI

XXVII

XXVIII

XXIX

XXX

XXXI

XXXII

XXXIII

In a fourth aspect, the invention provides a method for inhibitingbacterial growth, the method comprising administering an acyl phosphateor acyl phosphonate β-lactamase inhibitor. Preferably, such inhibitorshave the general mixed anhydride structure of Formula I:

or salt thereof;

wherein X is alkyl, aryl, aralkyl, or heterocyclic radical; Y is Z orOZ; and Z is alkyl, aryl, aralkyl, acyl, heterocyclic radical, orphosphonyl; provided that when Y is Z, then Z is not phosphonyl.

Preferred values for X, Y, and Z according to this aspect of theinvention are as described above for the first aspect.

In certain preferred embodiments, X and Y are taken together with theremaining atoms of the chain to form a cyclic structure of Formula II:

wherein Y is O or alkylene and X is alkylene, cycloalkylene, fusedheterocycle, heteroarylene, or arylene, and wherein the alkylene,cycloalkylene, fused heterocycle, heteroarylene, and arylene groups maybe optionally substituted. In certain preferred embodiments, X is afused carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. Inone such preferred embodiment, X is phenylene, giving the structure ofFormula III:

wherein Y is O or alkylene.

The methods according to this aspect of the invention are useful forinhibiting bacterial growth in a variety of contexts. For example, suchmethods can be used to prevent the growth of β-lactam resistant bacteriain experimental cell cultures. Such methods can also be used to preventthe growth of β-lactam resistant bacteria in veterinary contexts. Inaddition, such methods can be used to prevent the growth of β-lactamresistant bacteria in human patients.

Accordingly, in a preferred embodiment of this aspect, the inventionprovides methods for inhibiting bacterial growth in an animal, includinga human, comprising the step of administering a therapeuticallyeffective amount of β-lactamase inhibitors according to the inventionfor a therapeutically effective period of time to the animal.

The terms “therapeutically effective amount” and “therapeuticallyeffective period of time” are used to denote known treatments at dosagesand for periods of time effective to show a meaningful patient benefit,i.e., healing of conditions associated with bacterial infection, and/orbacterial drug resistance. The β-lactamase inhibitors of the inventionmay be administered to the animal by any route, including parenterally,orally, sublingually, transdermally, topically, intranasally,intratracheally, or intrarectally. When administered systemically, thetherapeutic composition is preferably administered at a sufficientdosage to attain a blood level of inhibitor from about 0.1 μg/mL toabout 1 mg/mL, more preferably from about 0.1 μg/mL to about 100 μg/mL,and most preferably from about 0.1 μg/mL to about 10 μg/mL. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. In apreferred embodiment, the inhibitor is administered orally.

In certain preferred embodiments of the method according to this aspectof the invention, a β-lactamase inhibitor according to the invention isco-administered with an antibiotic. In a particularly preferredembodiment of the invention, the co-administered antibiotic is aβ-lactam antibiotic. For purposes of this invention, the term“co-administered” is used to denote sequential or simultaneousadministration. In certain other preferred embodiments, the β-lactamaseinhibitor according to the invention has antibiotic activity, and thuseither can be administered alone or can be co-administered with aβ-lactam antibiotic or any other type of antibiotic. In someembodiments, more than one β-lactamase inhibitor may be administeredsequentially or simultaneously.

The following examples are intended to further illustrate certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention.

EXAMPLES Example 1 Chemical Synthesis of β-lactamase Inhibitors

Representative acyl phosphate and acyl phosphonate inhibitors havingstructures IV and VII-XV according to the present invention weresynthesized according to standard protocols. The following paragraphsset forth available synthetic approaches according to methods known inthe art.

The inhibitor of structure XIII (sodium benzoyl phenyl phosphate) wasprepared according to standard methods (e.g., by a modification of themethod employed by Jencks, et al. J. Biol. Chem. 234: 1272-1279 (1959))for the synthesis of acetyl phenyl phosphate. Thus, to a solution of 2.0g (7.8 mmol) of disodium phenyl phosphate (Adrich Chemical Co.) in 15 mLof water, cooled in an ice bath, was added 3.5 g (15.8 mmol) of benzoicanhydride (Acros Organics) dissolved in 10 mL pyridine, dropwise withstirring. After 25 min., the reaction mixture was extracted three timeswith diethyl ether and the aqueous phase then freeze-dried. The solidresidue was recrystallized twice from water and characterized by ¹H NMRspectra.

The inhibitor having structure XIV (sodium dibenzoyl phosphate) wasprepared in an analogous fashion to the inhibitor having structure XIII,beginning with disodium hydrogen phosphate and benzoic anhydride in a1:2 molar ratio. The product was recrystallized twice from water, withfinal melting point 195-197° C.

The inhibitors having structures X-XII were also prepared in ananalogous fashion to the inhibitor having structure XIII, and werecharacterized as follows:

Compound X: ¹H NMR (²H₂O): δ3.83 (s, 2H), 6.99 (d, J=8.3 Hz, 2H), 7.20(t, J=7.4 Hz, 1H), 7.3-7.5 (m, 7H); ³¹P (²H₂O): δ−15.2.

Compound XI: ¹H NMR (²H₂O): δ7.43 (d, J=8.7 Hz, 2H), 7.56 (t, J=7.9 Hz,2H), 7.74 (t, J=7.4 Hz, 1H), 8.07 (d, J=7.6 Hz, 2H), 8.29 (d, J=8.9 Hz,2H); ³¹P (²H₂O): δ−15.6.

Compound XII: ¹H NMR (²H₂O): δ7.41 (d, J=8 Hz, 1H), 7.51 (m, 4H), 7.67(m, 2H), 7.83(d, J=8 Hz, 1H),7.91 (d, J=8 Hz, 2H),8.02 (d, J=8 Hz, 2H).

The inhibitors having structures IV and XVII-XXXI were synthesized usingthe procedure of Laird and Spence, J. Chem. Soc. Perkin II, 1434-1436(1973). Thus, the silver salt of a phenyl, or substituted phenyl,phosphate or phosphonate (3.0 mmol) and benzoyl chloride or asubstituted benzoyl chloride (6.0 mmol) were stirred together inacetonitrile (15 mL) overnight under nitrogen at room temperature. Theresulting mixture was added dropwise into a stirred solution of sodiumbicarbonate (3.0 mmol) in H₂O (2.0 mL) at 0° C. Stirring was continuedfor 30-45 minutes at room temperature. More acetonitrile (50 mL) wasadded to the solution, which was then cooled for 1 hour at 0° C. Theprecipitate which appeared was collected and recrystallized from waterto give the pure sodium salt of the acyl phosphate or acyl phosphonate(55-60% yield). The compounds were characterized by ¹H NMR, ³¹P NMR, andIR.

Compound XVII: ¹H NMR (²H₂O): δ0.80 (t, J−6.9 Hz, 3H, CH₃), 1.18-1.30(m, 4H, Me(CH₂)₂), 1.36 (t, J=6.9 Hz, 2H, Me(CH₂)₂CH₂), 1.65 (quin,J=6.8 Hz, 2H), Me(CH₂)₃CH₂), 4.06 (dd, J=7.0, 6.7 Hz, 2 H, Me(CH₂)₄CH₂),7.57 (t, J=7.7 Hz, 2H, ArHCO), 7.73 (t, J=7.4 Hz, 1H, ArHCO), 8.09 (d,J=7.1 Hz, 2H, ArHCO); ³¹P (²H₂O) δ−9.04; ν_(max) (KBr) 1708s (C═O).

Compound XVIII: ¹H NMR (²H₂O): δ7.52 (t, J=7.4 Hz, 2H, ArHCO), 7.70 (t,J=7.4 Hz, 1H, ArHCO), 8.03 (d, J=8.3 Hz, 2H, ArHCO), 8.07 (d, J=8.3 Hz,2H, ArHP) 8.32 (d, J=8.7 Hz, 2H, ArHP); ³¹P (²H₂O) δ5.75; ν_(max) (KBr)1705s (C═O).

Compound XIX: ¹H NMR (²H₂O): δ7.52 (t, J=7.4 Hz, 2H, ArHCO), 7.63-7.77(m, 2H, ArHCO and ArHP), 8.01 (t, J=7.2 Hz, 2H, ArHCO), 8.22 (dd, J=12.9Hz, J=7.6 Hz, 1H, ArHP), 8.41 (d, J=8.3 Hz, 1H, ArHP); 8.67 (d, J=14.7Hz, 1H, ArHP); ³¹P (²H₂O) δ5.37; ν_(max) (KBr) 1706s (C═O).

Compound XX: ¹H NMR (²H₂O): δ7.44-7.61 (m, 5H0, 7.82-7.91 (m, 5H),7.95-7.98 (m, 1H, Naph), 8.33 (d, J=14.3 Hz, 1H, Naph); ³¹P (²H₂O)δ3.03; 1702s (C═O).

Compound XXI: ¹H NMR (²H₂O): δ7.39 (t, J=7.3 Hz, 1H, ArHCO), 7.46-7.52(m, 4H, ArHCO), 7.59 (s, 1H, biphen), 7.64 (dd, J=3.2, 8.3 Hz, 2H,biphen), 7.70 (d, J=7.2 Hz, 2H, biphen), 7.85 (d, J=8.2 Hz, 2H, PhArHP);7.90 (dd, J=7.0 Hz, 1.5 Hz, 2H, PhArHP); ³¹P (²H₂O) δ2.97; ν_(max) (KBr)1706s (C═O).

Compound XXII: ¹H NMR (²H₂O): δ0.79-0.87 (m, 3H, CH₃), 1.20-1.42 (m, 6H,Me(CH₂)₃), 1.50-1.63 (m, 2H, Me(CH₂)₃CH₂), 1.94 (quin, J=8.5, 17.0 Hz,2H, Me(CH₂)₄CH₂), 7.56 (t, J=7.7 Hz, 2H, ArHCO), 7.72 (t, J=7.4 Hz, 1H,ArHCO), 8.08 (d, J=8.1 Hz, 2H, ArHCO); ³¹P (²H₂O) δ25.97; ν_(max) (KBr)1700s (C═O).

Compound XXIII: ¹H NMR (²H₂O): δ7.21-7.30 (m, 3H, ArHOP), 7.41 (t, J=7.8hz, 2H, ArHOP), 8.24 (d, J=8.8 Hz, 2H, ArHCO), 8.36 (d, J=8.7 Hz, 2H,ArHCO); ³¹P (²H₂O) δ−14.63; ν_(max) (KBr) 1726s (C═O).

Compound XXIV: ¹H NMR (²H₂O): δ7.21-7.29 (m, 3H, ArHOP), 7.41 (t, J=7.8Hz, 2H, ArHOP), 7.78 (t, J=8.0 Hz, 1H, ArHCO), 8.41 (d, J=7.7 Hz, 1H,ArHCO), 8.53 (d, J=8.3 Hz, 1H, ArHCO), 8.85 (s, 1H, ArHCO); ³¹P (²H₂O)δ−14.59; ν_(max) (KBr) 1718s (C═O).

Compound XXV: ¹H NMR (²H₂O): δ6.55 (dd, J=2.2, 15.9 Hz, 1H, PhCH),7.21-7.29 (m, 3H, ArH), 7.42 (t, J=7.8 Hz, 2H, ArH), 7.46-7.60 (m, 3H,ArHOP), 7.64-7.69 (m, 2H, ArHOP), 7.82 (d, J=15.9 Hz, 1H, CHCO); ³¹P(²H₂O) δ−14.62; ν_(max) (KBr) 1701s (C═O).

Compound XXVI: ¹H NMR (²H₂O): δ7.22-7.30 (m, 3H, ArHOP), 7.42 (t, J=8.8Hz, 2H, ArHOP), 7.52 (t, J=7.4 Hz, 1H, ArHthiophene), 7.59 (t, J=7.2 Hz,1H, ArHthiophene), 7.98-8.04 (m, 2H, ArHthiophene), 8.24 (s, 1H,thiophene); ³¹P (²H₂O) δ−14.96; ν_(max) (KBr) 1705s (C═O).

Compound XXVII: ¹H NMR (²H₂O): δ7.0 (t, J=7.2 Hz, 1H), 7.16-7.22 (m,2H), 7.3 (t, J=7.8 Hz, 2H), 7.43 (t, J=7.2 Hz, 1H), 7.51 (t, J=7.3 Hz,2H), 7.75 (d, J=7.3 Hz, 2H), 7.81 (d, J=8.3 Hz, 2H), 7.96 (d, J=8.4 hz,2H); ³¹P (²H₂O) δ−14.34; ν_(max) (KBr) 1721s (C═O).

Compound XXVIII: ¹H NMR (²H₂O): δ2.29 (s, 6H, Ph(CH₃)₂CO), 7.15-7.26 (m,5H, ArHOP), 7.31-7.42 (m, 3H, ArHCO); ³¹P (²H₂O) δ−15.16; ν_(max) (KBr)1722s (C═O).

Compound XXIX:. ¹H NMR (²H₂O): δ3.91 (s, 3H, PhOCH₃), 7.08 (t, J=7.6 Hz,1H, ArHCO), 7.18-7.26 (m, 4H), 7.40 (t, J=8.0 Hz, 2H, ArHOP), 7.67 (t,J=8.9 Hz, 1H, ArHCO), 7.89 (dd, J=1.9, 8.0 Hz, 1H, ArHCO); ³¹P (²H₂O)δ−14.60; ν_(max) (KBr) 1718s (C═O).

Compound XXX: ¹H NMR (²H₂O): δ7.34-7.60 (m, 5H, benzo[b]thiophene), 7.71(s, 1 1H, Naph), 7.81-7.88 (m, 3H, Naph), 8.02 (d, J=7.3 Hz, 1H, Naph),8.07 (d, J=7.7 Hz, 1H, Naph), 8.13 (s, 1H, Naph); ³¹P (²H₂O) δ−14.97;ν_(max) (KBr) 1702s (C═O).

Compound XXXI: ¹H NMR (²H₂O): δ0.44 (d, J=8.5 Hz, 1H, Naph), 7.48-7.60(m, 4H), 7.67-7.73 (m, 2H), 7.86 (d, J=8.1 Hz, 1H, Naph), 7.91-7.97 (m,2H, Naph), 8.06 (d, J−8.2 Hz, 2H, ArHCO); ³¹P (²H₂O) δ−14.36; ν_(max)(KBr) 1711s (C═O).

The inhibitor of structure XXXII was prepared according to the methoddescribed by Kluger et al., Can. J. Chem. 74: 2395-2400 (1996). First,bis(tetraethylammonium)phenyl phosphate was prepared by the addition,with stirring over 10 min., of phenyl dichlorophosphate (7.86 g, 50mmol; Aldrich Chemical Co.) to 20 mL of water in an ice bath. Themixture was stirred for a further 1 hr. and the water removed by rotaryevaporation. Two equivalents of tetraethylammonium hydroxide (35% inwater, 41.1 mL; Aldrich) were then added, the pH adjusted to 7.0 withhydrochloric acid, and the resulting solution freeze-dried. The residue(16 g) was dissolved in 80 mL dichloromethane and dried overnight over 4Å molecular sieves.

Dicyclohexylcarbodiimide (1.2 g, 5.8 mmol) was added to a stirredsolution of phenylacetylglycine (1.16 g, 6 mmol) in dry dichloromethane(125 mL). The resulting mixture was stirred for 10 min., and thenbis(tetraethylammonium)phenyl phosphate (5 mmol) in dichloromethane wasadded. After a further hour, dicyclohexyl urea was removed by filtrationand the dichloromethane solution extracted twice with 50-mL portions ofwater. The aqueous solution was freeze-dried to yield the product whichwas then purified by Sephadex LH-20 chromatography in dichloromethane.The final oily product was characterized as a tetraethylammonium salt byits ¹H NMR spectrum [(D₂O) δ1.24 (t, 12H, NEt₄), 3.23 (q, 8H, NEt₄),3.65 (s, 2H, PhCH₂), 4.08 (s, 2H, NHCH₂), 7.3 (m, 10H, ArH)] and ESMA[m/e 608.7; bis(tetraethylammonium cation)].

The inhibitor of structure XXXIII was prepared as follows.N-(Benzyloxycarbonyl)aminomethylphosphonic acid was prepared asdescribed by Rahil and Pratt, J. Chem. Soc. Perkin Trans. 2, 947-950(1991). The required acyl phosphate was then obtained by the method ofJencks and Carriuolo, J. Biol. Chem. 234, 1272-1274 (1959), where theratio of starting materials, the time of mixing, and the mode of removalof pyridine, which catalyzes hydrolysis of the product as well as itsformation, were varied to optimize the yield. Thus, a solution ofbenzoic anhydride (0.94 g, 4.2 mmol; Acros Organics) in pyridine (0.5mL) was added dropwise with stirring to an ice-cooled solution ofN-(benzyloxycarbonyl)aminomethylphosphonic acid (100 mg, 0.41 mmol) andsodium hydroxide (0.85 mmol) in water (1.8 mL). After 15 min., thereaction mixture was rapidly extracted three times with diethyl ether,where suction by an aspirator was used to remove the ether phase. The pHof the aqueous phase was lowered to 3.5 by addition of 1 M HCl and theresulting solution freeze-dried. This procedure yielded a mixture ofproduct and starting phosphonate in a ratio of ca. 5:1. The productcould be isolated by precipitation as a N,N′-dibenzylethylenediaminesalt. Thus, the above product was dissolved in a minimum volume of waterand to it was added an equal volume of a 14 mM aqueous solution ofN,N′-dibenzylethylenediamine diacetate. The resulting mixture wasstirred in an ice bath for 10 min. and the precipitated product removedby filtration, washed with water, and dried in vacuo. The product, acolorless solid was characterized by its melting point (133-135° C.), ¹HNMR spectrum [(²H₆-DMSO) δ3.41 (s, 4H, CH₂CH₂), 3.60 (dd, J=6, 12 Hz,CH₂P), 4.15 (s, 4H, CH₂Ph), 5.11 (s, 2H, CH₂O), 7.10 (br t, 1H, NH), 7.5(m, 20H, ArH)], ³¹P NMR spectrum [(²H₆-DMSO) δ10.5], IR spectrum [(KBr)γ_(C═O)=1709 cm⁻¹], and ESMS (m/e 348.1; M+H⁺).

The representative cyclic acyl phosphate and cyclic acyl phosphonateinhibitors having structures V, VI, and XVI according to the presentinvention were synthesized according to standard protocols. Thefollowing paragraphs set forth possible synthetic approaches, accordingto methods known in the art.

The acyl phosphate1-hydroxy-4.5-benz-2.6-dioxaphosphorinanone-(3)-1-oxide were synthesizedby the hydrolysis of1-chloro-4.5-benz-2.6-dioxaphosphorinanone-(3)-1-oxide according tostandard methods (e.g., Bruice et al., J. Am. Chem. Soc. 117:12064-12069(1995). Salicylic acid (69.0 g) and phosphorus oxychloride (76.7 g) weregradually heated to 150° C. for 2 hours. The viscous product wasdistilled and the fraction b.p. 116-125° C./0.02 mm. collected andcrystallized from carbon tetrachloride according to standard methods(e.g., Montgomery et al. J Chem Soc 4603-4606 (1956)).

After a preparative scale hydrolysis, the inhibitor of structure V wasisolated and characterized as a stable dicyclohexylamine salt. Thecyclic phosphoryl chloride (500 mg; 2.3 mmol) was suspended in 60 mL ofanhydrous acetonitrile (Aldrich, Sure/Seal) and water (41 μL, 2.3 mmol)added with magnetic stirring. The mixture was then gently warmed untilthe crystals of the starting material disappeared (ca. 5 min.). Twomolar equivalents of redistilled dicyclohexylamine (913 μL) were thenadded. The immediately precipitating solid (cyclohexylaminehydrochloride) was removed by vacuum filtration and the remainingsolvent by rotary evaporation. The residual colorless solid wasrecrystallized from acetonitrile/diethyl ether yielding 310 mg (35%) ofthe required product. (Characterization: mp 160-163° C.; ¹H NMR (²H₂O):δ(aromatics) 7.27 (d, 1H, J=8 Hz), 7.35 (t, 1H, J=8 Hz), 7.78 (t, 1H,J=8 Hz), 8.05 (d, 1H, J=8 Hz); ³¹P (₂H₂O) δ−12.55; UV (20 mM MOPSbuffer, pH 7.5) λ_(max)239 nm (ε=1.01×10⁴ cm⁻¹M⁻¹), 295 nm (ε=2.52×10³cm⁻¹M⁻¹); IR (KBr) 1737 cm⁻¹ (C═O), 1613 cm⁻¹ (C═C), 1286 cm⁻¹ (P═O);Anal. Calcd. for C₁₉H₂₈NO₅P: C, 59.80; H, 7.40; N, 3.67; P, 8.12. Found:C, 60.11; H, 7.44; N, 3.70; P, 7.91.). The cyclic phosphate hydrolyzedslowly in buffer at neutral pH. The rate ofhydrolysis—k_(obs)=1.7×10⁻⁵s⁻¹ in 20 mM MOPS, pH 7.5, 25° C.—is inaccord with literature values. Kintetics were determinedspectrophotometrically at 240 nm, and an isosbestic point for thehydrolysis of salicyl phosphate to salicylate was observed.

The cyclic acyl phosphate inhibitor having structure XVI wassynthesiszed according to the published procedure (Marecek and Griffith,J. Am. Chem. Soc. 92:917 (1970)).

The representative cyclic acyl phosphonate inhibitor having structure VIwas synthesized by thermal hydration of the corresponding acyclic diacidby heating up to 150° C.

Example 2 In Vitro Inhibition of β-Lactamase and of DD-Peptidase

To evaluate the inhibitors and the methods of the invention,representative inhibitors preparations were tested using class A (TEM-2plasmid TEM)) β-lactamase and class C (Enterobacter cloacae P99β-lactamase) β-lactamases (Porton Down, Wiltshire, U.K.) as well asrepresentative DD-peptidases/carboxydases (e.g., Streptomyces R61DD-peptidases) as the assayed enzyme.

Steady state kinetic parameters were directly obtained forrepresentative inhibitors with the P99 β-lactamase with TEM β-lactamaseby standard methods (e.g., Wilkinson, Biochem J. 80:324-332 (1989)) fromspectrophotometric initial velocity measurements. Kinetic parameterswere also obtained for DD-peptidase as the assayed enzyme, according tostandard methods using hyppuryl-DL-phenyl-lactate (a known DD-peptidasesubstrate) (e.g. Govardhan and Pratt, Biochemistry 26:3385-3395 (1987)).The concentration of stock enzyme solutions were determinedspectrophotometrically (e.g., Xu, et al., Biochemistry 35:3595-33603(1996)).

Representative class C and a class A β-lactamases were inhibited byrepresentative inhibitors of Formulae I and II in a time-dependentfashion as described by the relation:

where k_(i) (s⁻¹M⁻¹) represents the second order rate constant forinhibition of assayed enzymes upon treatment with the inhibitorsaccording to the present invention.

Representative inhibitors of the invention having Formula I may act byforming a covalent bond between β-lactamases and the inhibitor (i.e.,irreversible inhibition). This type of inhibition is therefore timedependent. It is distinguished from reversible inhibition in so far thatthe backward reaction is extremely slow. Kinetically, irreversibleinhibition is inhibition in which the inhibitor does not significantlydissociate from the enzyme within the experimental time frame. Thus,irreversible inhibition is measured by how fast the event takes place(rate constant k_(i)). Thus, inhibitory efficiency is expressed by largek_(i) values.

Representative inhibitors of Formula I may also act by forming atransient acyl-enzyme intermediate. Inhibition results from a slow turnover (de-acylation) of the intermediate. Hence, inhibition is by thesecond order rate constant for the formation of the acyl-enzymeintermediate (i.e., k_(cat)/K_(m)). Notably, an inhibitor according tothe present invention may inhibit according to both mechanisms asillustrated for example by the inhibitor having structure VII, for whichboth k_(cat)/K_(m) and k_(i) values are reported.

Representative β-lactamase inhibition test results for inhibitors of theinvention are shown in Table 3. In Table 3, k_(cat)/k_(m) values arepresented in plain text, with k_(i) values for representative inhibitorsappearing in parentheses. The data in Table 3 also show that someinhibitors of the invention (e.g., inhibitors having structures V andVII) may have antibiotic properties, as evidenced by their inhibition ofDD-peptidase.

TABLE 3 k_(cat)/k_(m) × 10⁻⁴ (s⁻¹ M⁻¹) and k₁ (s⁻¹ M⁻¹) Enterobactercloacae TEM-2 No. Inhibitor Structure P99 β-lactamase plasmid (TEM)DD-peptidase IV

0.690 (70) 0.0002 (1.6) V

0.560 0.0014 0.46 VII

0.0067 (6.0) (0.2) X

3.10 (≦0.04) XI

0.644 (≦2) XIII

0.607 (−) 0.001 (0.2) XIV

0.395 (−) 0.120 (14.3) XVII

0.125 (≦1) XVIII

0.0125 (25 ± 3) XIX

0.0179 (65 ± 19) XX

0.0551 (115 ± 5) XXI

0.117 (89 ± 4) XXII

0.0226 (11.1 ± 0.1) XXIII

6.27 (700 ± 100) XXIV

7.88 (440 ± 60) XXV

3.04 (≦1) XXVI

23.2 (≦5) XXVII

6.83 (≦3) XXVIII

(20.9 ± 1.5) XXIX

0.0562 (3.6 ± 0.4) XXX

131 (1813 ± 3) XXXI

1.75 (≦1) XXXII

100 (−) 0.188 (−) 2.05 (−) XXXIII

— (5.9 ± 0.3 × 10⁴) — (130 ± 8) 1.65 ± 0.13 (0.005) positive clavulanicacid 4 4.8 × 10⁴ control

Example 3 Inhibition of P99 β-lactamase

The enzyme (Enterobacter cloacae P99 β-lactamase) was dissolved to aconcentration of 1 mg/mL in 20 mM MOPS buffer at pH 7.50, and thendiluted 200-fold in the same buffer containing 0.1% BSA. Thecommercially available substrate nitrocefin was added as a 100 μM stocksolution in 20 mM MOPS buffer, and inhibitors were also added assolutions in MOPS buffer. In a typical assay the following amounts wereused: 300 μL of nitrocefin, 10 μL of P99 β-lactamase, x μL of inhibitor,and 110x μL of MOPS buffer. Enzyme activity was measured at 25 EC, andthe reaction was monitored spectrophotometrically at 482 nm. IC₅₀ valueswere determined from the dose-response plots of the initial reactionrate vs. inhibitor concentration. Results are presented in Table 4.

TABLE 4 IC₅₀ (μM) P99 No. Inhibitor Structure β-lactamase X

30 XI

39 XII

 8

Example 4 Microbiological Testing

To test the inhibitors of the invention, a wide range of organisms areused to assess compounds according to the invention both as antibioticsand as β-Lactamase inhibitors: Initial screening for antibiotic activityutilizes penicillin sensitive S. aureus [ATTC# 25923], ampicillinsensitive H. influenzae [ATCC#10211] and clinical laboratory isolates ofampicillin sensitive E. faecalis and H. influenzae, and non-resistant P.aeruginosa. Initial studies utilize, in microtiter wells, 1 microgram ofcompound in 10 microliters diluted stock solution, 41,100 CFU ofbacteria in 10 microliters broth solution and 80 microliters CAMHB. Thecount verification well contains 10 microliters CAMHB in place of thetest compound. Tests for β-Lactamase inhibition utilize 1:1 and 1:10wt/wt ratios of β-Lactam/β-Lactamase inhibitor (10 micrograms/mLβ-Lactam) and initially utilize penicillinase-producing S. aureus [ATTC#29213] with piperacillin, extended-spectrum lactamase-producingKlebsiella oxytoca [ATCC# 51983] with piperacillin or ticarcillin,cephalosporinase-producing E. cloace [ACC# 23355] with cefoxitin orceftriaxone, broad-spectrum lactamase-producing E. coli [ATCC# 35218]with amoxicillin, piperacillin or ticarcillin, broad-spectrumlactamase-producing N. gonnorhea [ATTC# 49226] with amoxicillin,lactamase-producing H. influenzae [ATTC# 43163] with tricarcillin, andlactamase-producing E. faecalis [ATTC# 49757] with amoxicillin.Microbiological and enzymatic testing is carried on duplicate plateswith negative control plates.

Bacterial susceptibility may also be assayed by the gradient plate(i.e., the Kirby-Bauer) method. (Microbiology: Including Immunology andMolecular Genetics (3rd ed.) (Davis et al. eds. (1980) J.P. LippincottCo., Philadelphia, Pa.). Briefly, according to this method standardfilter paper disks impregnated with fixed amounts of compositions ofinhibitors, ranging from 100 μg/mL to 1 mg/mL and from 10 μg/mL to 100μg/mL and of inhibitors as well as antibiotic are dried. A “lawn” ofbacteria is seeded on the surface of an agar LB plate (see e.g.,Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory Press(1989)) in the presence of inhibitors concentrations with a swabmoistened with a standardized liquid culture, and several differentinhibitor compositions-impregnated on disks are placed on the surface ofthe agar. The plates are then incubated over-night and the diameter ofthe zone showing no growth is then measured. The diameter of this zoneof inhibition will be used to determine the minimal inhibitoryconcentration (MIC) value for susceptibility of the organism underscrutiny has high, intermediate, or inadequate insensitivity to the drugtested. These assays are expected to show that the inhibitors of theinvention are effective to render infecting microorganisms susceptibleand thus that the compositions and methods of the invention are usefulfor overcoming bacterial antibiotic resistance.

Broth or agar dilution methods are also used to determine the MIC valuefor susceptibility of bacterial isolates to the inhibitors of theinvention. Briefly described, tubes are inoculated to contain 10⁴ to 10⁸organisms/mL or LB plates (supra) are inoculated with 10³ to 10⁴organisms. Various concentrations of inhibitor compositions (and ofclavulanic acid, as positive controls) are added to several individualplates or tubes, (no inhibitor is added to negative control plates ortubes). These assays are expected to identify MIC ranges comparable tothose observed for clavulanic acid (see e.g., Bateson et al., Bioorg. &Med. Chem. Letts. 4: 1667-1672 (1994)).

Results of microbiological testing for compounds of the invention arepresented in Table 5. Compounds were tested at a concentration of 100μg/mL. Compounds not shown in the Table gave negative results in theseassays.

In Table 5, the microorganism tested against is shown in italics, whilethe antibiotic agent used is shown in plain text. The symbol (+) meansthat the inhibitor of the invention showed biological activity, asdetermined by an increase in antibiotic activity of the antibioticagent. When determined, the number in parentheses indicates the foldreduction in the MIC of the antibiotic agent when measured in thepresence of the inhibitor of the invention. The symbol (−) means thatthe inhibitor of the invention showed no biological activity, i.e., theantibiotic efficacy of the antibiotic agent was unchanged in thepresence of the inhibitor.

TABLE 5 Haemophilus Ent. Cloacae Staph. Aureaus Influenzae CompoundCeftriaxone Cefoxitin Piperacillin Ticarcillin IV 2 2 — — V — — 2 — VII2 2 — 0 VIII 2 2 — 0 XVI — — 2 —

Successful inhibition of bacterial β-Lactamase activity in these assaysis expected to be predictive of success in animals and humans.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

What is claimed is:
 1. A β-lactamase inhibitor of Formula I:

or a salt thereof; wherein X is alkyl, aryl, aralkyl, or heterocyclicradical; Y is Z or OZ; and Z is selected from the group consisting ofC₁₋₆ alkyl, phenyl, and naphthyl, any of which groups may be optionallysubstituted.
 2. A β-lactamase inhibitor of Formula I:

or a salt thereof; wherein X is alkyl, aryl, aralkyl, or heterocyclicradical; Y is Z, wherein Z is selected from the group consisting of C₁₋₆alkyl, phenyl, and naphthyl, wherein the phenyl or naphthyl isunsubstituted or is substituted with one or two substituentsindependently selected from the group consisting of C₁₋₄ alkyl, C₁₋₄alkoxy, C₆₋₁₀ aryl, and nitro.
 3. A β-lactamase inhibitor selected fromthe group consisting of:


4. A β-lactamase inhibitor selected from the group consisting of:


5. A β-lactamase inhibitor selected from the group consisting of:


6. A pharmaceutical composition, comprising a β-lactamase inhibitorselected from the group consisting of:

a pharmaceutically acceptable carrier, diluent, or excipient.
 7. Apharmaceutical composition, comprising a β-lactamase inhibitor selectedfrom the group consisting of:

and salts thereof; and a pharmaceutically acceptable carrier, diluent,or excipient.
 8. A pharmaceutical composition, comprising a β-lactamaseinhibitor selected from the group consisting of:

and salts thereof; and a pharmaceutically acceptable carrier, diluent,or excipient.
 9. The composition according to any of claims, 6, 7, or 8further comprising an antibiotic agent.
 10. The composition according toclaim 9, wherein the antibiotic agent is a β-lactam antibiotic.
 11. Amethod for inhibiting β-lactamase activity, comprising administering aβ-lactamase inhibitor selected from the group consisting of:


12. A method for inhibiting β-lactamase activity, comprisingadministering a β-lactamase inhibitor selected from the group consistingof:

and salts thereof.
 13. A method for inhibiting β-lactamase activity,comprising administering a β-lactamase inhibitor selected from the groupconsisting of:

and salts thereof.
 14. A method for inhibiting bacterial growth,comprising administering a β-lactamase inhibitor selected from the groupconsisting of:


15. A method for inhibiting bacterial growth, comprising administering aβ-lactamase inhibitor selected from the group consisting of:

and salts thereof.
 16. A method for inhibiting bacterial growth,comprising administering a β-lactamase inhibitor selected from the groupconsisting of:

and salts thereof.
 17. The method according to claim 15 or 16, furthercomprising administering an antibiotic.
 18. The method according toclaim 17, wherein the antibiotic agent is a β-lactam antibiotic.